Hyperinsulinemia contributes to cardiovascular dysfunction in obesity and diabetes through central actions to increase sympathetic nerve activity (SNA) and alter baroreflex function. The hypothalamic arcuate nucleus (ARC) expresses a high abundance of insulin receptors, and direct injection of insulin into the ARC increases SNA. Therefore, the present study tested the hypothesis that circulating insulin acts on ARC neurons to increase SNA by using an anti-insulin Affibody to locally neutralize insulin within the ARC. The anti-insulin Affibody versus anti-IgG completely abolished the increased lumbar SNA to ARC injection of insulin (P<0.05). Blood pressure and heart rate were not different between groups. In a second set of experiments, pre-treatment with ARC injection of the anti-insulin Affibody versus control Affibody also prevented the increase in lumbar SNA during a hyperinsulinemic-euglycemic clamp (P<0.05).